Nitric oxide (NO) has been shown to be an important biological second messenger which is biosynthesized by a family of enzymes called nitric oxide synthases (EC 1.14.13.39). Three principal isoforms of this enzyme have been isolated and characterized, each associated with different physiological functions. (Kerwin, J. F.; Lancaster, J. R., J. R. Jr.; Feldman, P. L., J. Med. Chem. 1995, 38, 432-62.) As is well known in the art, there are numerous isoforms of NOS. Those isoforms are designated herein as endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). The isoform from endothelial cells (eNOS) is involved in the regulation of smooth muscle relaxation, blood pressure lowering, and inhibition of platelet aggregation. Neuronal nitric oxide synthase (nNOS) is important for long-term potentiation, and an inducible form (iNOS), which acts in host defense, is generated by activated macrophage cells during an immune response. The endothelial and neuronal isoforms are expressed constitutively and require Ca.sup.2+ and calmodulin for activity; the macrophage form is induced by cytokines or bacterial lipopolysaccharides and is Ca.sup.2+ and calmodulin independent. All forms of the enzyme require NADPH, tetrahydrobiopterin, heme, and at least FAD if not both FAD and FMN.
Despite the extraordinary importance of NO to the apparent health of organisms, it also has been associated with a large number of harmful effects as a result of its reactive free radical properties; in fact, NO has a lifetime of only a few seconds at physiological pH and temperature. Overproduction of NO, consequently, has been implicated in a wide variety of diseases (Kerwin, J. F.;
Heller, M. The Arginine-Nitric Oxide Pathway: A Target for New Drugs. Med Res. Rev. 1994, 14,23-74). NO overproduction by nNOS has been implicated in strokes, septic shock, seizures, schizophrenia, migraine headaches, Alzheimer's disease, long-term depression, and priapism. (Choi, D. W.; Rothman Annu. Rev. Neurosci, 1990, 13, 171-182, Garthwaite, J. In the NMDA Receptor; Watkins, J. C. Collingridge G. L. Ed.: Oxford University Press; Oxford England; 1989, pp. 187-205; Crossin, K. L. Trends Biochem. Sci, 1991, 16, 81-2; Kerwin, J. F.; Lancaster, J. R., J. R. Jr.: Feldmand, P. L. 1995,38,432-62. J. Med. Chem; Das, I.; Khan, N. S.; Puri, B. K.; Sooranna, S. R.; de Belleroche, J.; Hirsch, S. R. Biochem. Biophys. Res. Commun. 1995, 212, 375-380.; Thomsen, L. L.; Iversen, H. K.; Lassen, L. H.; Olesen, J. CYS Drugs 1994, 2, 417-22.; Dorheim, M. A.; Tracey, W. R.; Pollock, J. S.; Grammas, P. Biochem. Biophys. Res. Commun. 1994,205, 659-665.; Shibuki, K.; Okada, D. Nature 1991, 349, 326-328.; Burnett, A. L.; Lowenstein, C. J.; Bredt, D.S.; Chang, T. S. K.; Snyder, S. H. Science 1992,257,401-403). iNOS overproduction of NO has been associated with tolerance to and dependence on morphine, development of colitis, tissue damage and inflammation, overproduction of osteoclasts, leading to osteoporosis, Paget's disease, rheumatoid arthritis, and destruction of photoreceptors in the retina. (Bhargava, H. N. Gen. Pharmacol. 1995, 26, 1049-1053).; Seo, H. G.; Takata, I.; Nakamura, M.; Tatsumi, H.; Suzuki, K.; Fujii, J.; Taniguchi, N. Arch. Biochem. Biophys. 1995, 324, 41-47., Kubes, P.; Suzuki, M.; Granger, D. N. Proc. Natl. Acad. Sci. USA 1991, 88, 4651-4655; MacIntyre, I.; Zaidi, M.; Towhidul Alam, A. S. M.; Datta, H. K.; Moonga, B. S.; Lidbury, P. S.; Hecker, M.; Vane, J. R. Proc. Natl. Acad. Sci. USA 1991, 88,2936-2940; and Ross, C. A.; Bredt, A.; Snyder, S. H. Trends Neurosci. 1990, 13, 216-22. This suggests that inhibition of NOS would have a significant beneficial effect on disease states arising from the overproduction of NO.
A wide variety of compounds have been shown to inhibit NOS, (Marlett, M. A. J. Med. Chem. 1994, 37, 1899-1907; Moore, W. M.: Webber, R. K. Fok, K. F.; Jerone, G. M.: Kornmeier, C. M.; Tjoeng, F. S.; Currie, M. G. Bioorg. Med. Chem 1996. 4. 1559-1564). However, because of the general importance of NO to human health, selective inhibition of the isoforms of NOS is essential. Selectivity for the three isoforms already has been reported to some degree. The early inhibitors were analogs of L-arginine. N.sup..omega. -Methyl-L-arginine and N.sup..omega. --ethyl-L-arginine, however, show only about a factor of 2 selectivity; N.sup..omega. --methyl-L-arginine is selective for eNOS and nNOS over iNOS and N.sup..omega. --ethyl-L-arginine is selective for iNOS over nNOS and eNOS. (Moore, W. M.; Webber, R. K.; Fok, K. F.; Jerone, G. M.; Connor, J. R.; Manning, P. T.; Wyatt, P. S.; Misko, R. P.; Tjoeng, F. S.; Currie, M. G. J. Med. Chem. 1996. 39. 669-72). N.sup..omega. --Nitro-L-arginine is about 300-fold selective for nNOS over iNOS (Furfine, E. S.; Harmon, M. F.; Paith, J. E.; Garvey, E. P. Biochemistry, 1993, 32, 8512-8517). 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and S-ethylisothiourea were termed "potent and selective inhibitors,: but had only 10-40 fold selectivity in favor of iNOS (Furfine, E. S.; Harmon, M. F.; Paith, J. E.; Garvey, E. P. Biochemistry, 1993, 32, 8512-8517; Nakane, M.; Klinghofer, V.; Kuk, J. E.; Donnelly, J. L.; Budzik, G. P.; Pollock, J. S.; Basha, F.; Carter, G. W. Mol. Pharmacol. 1995, 47, 831-4. 2-Iminoazaheterocycles showed selectivities of 1.1-9 in favor in iNOS over eNOS and nNOS (Moore, W. M.; Webber, R. K.; Fok, K. F.; Jerone, G. M.; Connor, J. R.; Manning, P. T.; Wyatt P. S.; Misko, R. P.; Tjoeng, F. S.; Currie, M. G. J. Med. Chem. 1996, 39, 669-72). Various indazole analogs had selectivities of 5-10 for either nNOS or iNOS. (Wolff, D. J.; Grivin, B. J. Arch. Biochem. Biophys. 1994, 311, 300-306). Imidazole analogs exhibited selectivities in the range of 3-6 fold in favor of iNOS; (Wolff, D. J.; Gribin, B. J. Arch. Biochem. Biophys. 1994, 311, 293-9). Aminoguanidine shows a 50-fold selectivity for iNOS over nNOS and 500-fold over eNOS (Wolff, D. J.; Lubeskie, Arch. Biochem. Biophys. 1995, 316, 290-301). Several "potent and selective" series of isothiourea analogs favored inhibition of iNOS over eNOS by factors of between 2- and 6-fold with one analog being 19-fold selective; bisisothioureas were more selective with one analog showing a selectivity of 190-fold in preference of iNOS, (Wolff, D. J.; Lubeskie,. Arch. Biochem. Biophys. 1995, 316, 290-301). S-Methyl- and S-ethyl-L-thiocitrulline, also called "potent and selective," were 10- and 50-fold, respectively, more selective for nNOS than eNOS (Furfine, E. S.; Harmon, M. F.;. Paith, J. E.; Knowles, R. G.; Salter, M.; Kiff, R. J.; Duffy, C.; Hazelwood, R.; Oplinger, J. A.; Garvey, E. P. J. Biol. Chem. 1994, 269, 26677-83), L-N.sup.6 -(1-Iminoethyl)lysine, another "selective" inhibitor of NOS favors the inhibition of iNOS by a factor of 30 over eNOS and by 13 over nNOS (Moore, W. M.; Webber, R. K.; Jerone, G. M.; Tjoeng, F. S.; Misko,. T. P.; Currie, M. G. , J. Med. Chem. 1994, 37, 3886-8).
Because of the relatively high selectivity of N.sup..omega. --nitro-L-arginine for nNOS over iNOS, and the observation that it is a time-dependent inhibitor of nNOS, but a reversible inhibitor of iNOS (Dwyer, M. A.; Bredt, D. S.; Snyder, S. H. Biochem. Biophys. Res. Commun. 1991, 176, 113641) it was decided to determine if N.sup..omega. --nitroarginine-containing dipeptide esters or amides would have increased selectively for nNOS. Since L-arginine methyl ester, L-argininamide, and L-arginine-containing dipeptides are substrates for NOS it was thought that the dipeptide esters or amides may be functional as well. Furthermore, the fact that N.sup..omega. --nitro-L-arginine inhibits nNOS in vivo following intraperitioneal injection suggests that it crosses the blood-brain barrier, even though L-arginine poorly crosses (Hecker, M.; Walsh D. T.; Vance, J. R. On the substrate specificity of nitric oxide synthase. FEBS Lett. 1991, 294, 2214). This may be the result of the decrease in pKa of the guanidino group because of nitro substitution.